Lipopeptide inhibitors of ras oncoproteins

ABSTRACT

A method of inhibiting Ras activity in a cell comprising introducing a peptide or peptidomimetic into the cell, wherein the peptide or peptidomimetic is derived from or based upon the amino acid sequence of the C-terminal α-helix or hypervariable region (HVR) or a Ras protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a divisional of U.S. patent application Ser.No. 14/119,596, filed Jan. 30, 2014, which is a U.S. National Phase ofInternational Patent Application No. PCT/US2012/039623, filed May 25,2012, which claims the benefit of U.S. Provisional Patent ApplicationNo. 61/489,919, filed May 25, 2011, each of which is incorporated byreference in its entirety herein.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 36,556 Byte ASCII (Text) file named“722500_ST25.TXT,” dated Jan. 21, 2016.

BACKGROUND OF THE INVENTION

Ras proteins are small GTPases that act as signal transducers betweencell surface receptors and several intracellular signaling cascades.These molecules regulate such essential cellular functions as cellsurvival, proliferation, motility, and cytoskeletal organization (seeKarnoub et al., Nat. Rev. Mol. Cell Biol., 9: 517-531 (2008)).

Ras proteins function as GDP/GTP-regulated binary switches in signaltransduction cascades that can lead to cell growth, proliferation,differentiation, or survival. In its active form, Ras is bound to GTP.This causes a conformational change that allows it to interact and bindto several effector molecules, most notably the members of the Raffamily, the RalGDS family, and Phosphoinositide 3-kinases (PI3 Kinase).Ras then cleaves GTP to GDP resulting in its inactivation. In itsoncogenic, mutated state, Ras is unable to hydrolyze GTP to GDP, thusstaying in an active state and activating numerous pathways.

The Ras superfamily has at least five major branches that include Ras,Rho, Ran, Arf/Sar, Rab. The four classical p21 Ras proteins are H-Ras(Harvey sarcoma viral oncogene), N-Ras (neuroblastoma oncogene), and thesplice variants K-Ras4A and K-Ras4B (Kirsten sarcoma viral oncogene)(see Karnoub et al., supra). They are collectively referred to as Ras.

The p21 Ras proteins share 85% of sequence homology and activate verysimilar signaling pathways. However, recent studies clearly demonstratethat each Ras isoform functions in a unique, radically different wayfrom the other Ras proteins in normal physiological processes as well asin pathogenesis (Quinlan et al., Future Oncol., 5: 105-116 (2009)).According to Catalogue of Somatic Mutations in Cancer(www.sanger.ac.uk/genetics/CGP/cosmic/), K-Ras mutations were detectedin 22.1% of analyzed human tumors, N-Ras in 8.2%, and H-Ras in 3.3%.

Mutations in cellular Ras have been found to be present in a largepercentage of all human cancers, such as leukemias, colon cancers, andlung cancer (see Quinlan et al., supra, and Boissel et al., Leukemia,20: 965-970 (2006)). More specifically, K-Ras mutations occur frequentlyin lung, pancreatic, and colon cancers, where as H-Ras mutations areprevalent in bladder, kidney, thyroid, and salivary gland cancers (seeShulz, Int. J. Cancer, 119: 1513-1518 (2006), and Yoo et al., Arch.Pathol. Lab Med., 124: 836-839 (2000)), and N-Ras mutations areassociated with myeloid malignancies, germ cell tumors, melanoma,hepatocellular carcinoma, and leukemia. Additionally, K-Ras mutation ispredictive of response to EGFR antagonists therapy in colorectal cancer(see Lopez-Chavez et al., Curr. Opin. Investig. Drugs, 10: 1305-1314(2009)).

Despite the central role of ras proteins in oncogenesis and wide-spreadefforts to develop ras-directed anti-cancer therapeutics, no selective,specific inhibitor of the ras pathway is available for clinical use, andras mutant cancers remain among the most refractory to availabletreatments (Adjei, J. Thorac. Oncol., 3: S160-163 (2008); Graham et al.,Recent Results Cancer Res., 172: 125-153 (2007); and Saxena et al.,Cancer Invest., 26: 948-955 (2008)). Moreover, no inhibitors actingdirectly on ras oncogenes have been developed even for in vitro use.Consequently, ras proteins are considered to be non-druggable targets.Therefore, there is a desire to identify inhibitors of ras oncoproteins.

BRIEF SUMMARY OF THE INVENTION

The invention provides a peptide or peptidomimetic comprising the aminoacid sequence comprising eight or more contiguous amino acids of theC-terminal α-helix of a Ras protein (e.g., eight or more contiguousamino acids of one of SEQ ID NOs: 66-68, or inverse thereof), whereinthe peptide or peptidomimetic comprises a total of about 30 or feweramino acids. In a related aspect, the invention provides a peptide orpeptidomimetic comprising X₁YTLVRX₂X₃RX₄X₅ (SEQ ID NO: 5) or the inversethereof, wherein the peptide or peptidomimetic comprises about 30 orfewer amino acids.

The invention also provides a peptide or peptidomimetic comprising fiveor more contiguous amino acids of the hypervariable region (HVR) of aRas protein (e.g., five or more contiguous amino acids of one of SEQ IDNOs: 69-72, or inverse thereof), wherein the peptide or peptidomimeticcomprises a total of about 30 or fewer amino acids. In a related aspect,the invention provides a peptide or peptidomimetic comprising the aminoacid sequence KTPGX₁VKIKK (SEQ ID NO: 6) or inverse thereof, the aminoacid sequence KKSKTK (SEQ ID NO: 7) or the inverse thereof, the aminoacid sequence SGPGX₁X₂SX₃X₄ (SEQ ID NO: 8) or the inverse thereof, orthe amino acid sequence GTQGX₁X₂GLP (SEQ ID NO: 9) or the inversethereof, wherein the peptide or peptidomimetic comprises about 30 orfewer amino acids.

The invention further provides a method of inhibiting the activity of aRas protein in a cell comprising introducing a peptide or peptidomimeticdescribed herein into the cell, whereby the activity of the Ras proteinis inhibited.

The invention also provides a method for inhibiting the growth orproliferation of a cancer cell comprising administering a peptide orpeptidomimetic described herein to the cancer cell, whereupon the growthor proliferation of the cancer cell is inhibited.

In addition, the invention provides a method of treating cancer in ahost comprising administering to the host a peptide or peptidomimeticdescribed herein or nucleic acid encoding same, whereby the cancer istreated.

Related compounds, compositions, and methods also are provided, as willbe apparent from the detailed description of the invention.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 depicts the primary structure alignment of four human Rasproteins with secondary structure elements shown above the sequences.The helix 6 and C-terminal hypervariable region (HVR) are shaded.Positions of the sequences that are conserved are marked with anasterisk. Positions of the sequences showing variation are marked withdots indicating the relative similarity between the residues that occupya given position in the sequence. Two dots below a given position in thesequence indicate substitution by more closely related residues and onedot or no dots indicates substitution by less similar residues.

FIG. 2 is a graph illustrating growth inhibition in human lung tumorcell lines (A549, H460, and H1944) and a mouse astrocytoma cell line(187436 tu2) expressing mutated K-Ras by treatment with a Ras inhibitor(KR-H6-23). The concentration (nM) of inhibitor is represented on thex-axis and the cell number (percent of control) is represented on they-axis.

FIG. 3 is a graph illustrating growth inhibition in human lung tumorcell lines (A549, H460, and H1944) and a mouse astrocytoma cell line(187436 tu2) expressing mutated K-Ras by treatment with a Ras inhibitor(KR-4A-4). The concentration (nM) of inhibitor is represented on thex-axis and the cell number (percent of control) is represented on they-axis.

FIG. 4 is a graph illustrating growth inhibition in a breast epithelialcell line (BJ-5ta, immortalized by transfection of normal cells withhuman Telomerase Reverse Transcriptase (hTERT)-expressing plasmid) and ahuman lung tumor cell line (A549) by treatment with a Ras inhibitor(kR-H6-30). The concentration (nM) of inhibitor is represented on thex-axis and the cell number (percent of control) is represented on they-axis.

FIG. 5 is a graph illustrating the inhibition of HCC15 cell motility bytreatment with Ras inhibitors (kR-4A-1 and kR-4B-1). The average closurein μm is indicated on the y-axis for control.

FIG. 6 is a graph illustrating the inhibition of NCC15 cell migration bytreatment with Ras inhibitors (kR-4A-1 and kR-4B-1). The average numberof cells migrated is indicated on the y-axis.

FIG. 7 is a graph illustrating the inhibition of HCC15 cell invasion bytreatment with Ras inhibitors (kR-4A-1 and kR-4B-1). The average numberof cells migrated is indicated on the y-axis.

FIG. 8 is a graph illustrating the suppression of growth of an H358human tumor in nude mice. Tumor volume change (mm³) for control (DMSOvehicle) (0) or 10 mg/kg kR-4B-8 treated (B8) (□) mice is indicated onthe y-axis. The number of days is indicated on the x-axis.

FIG. 9 is a graph illustrating the effect of a Ras inhibitor (kR-H6-48)on Lewis Lung Carcinoma isograft growth in female mice. Tumor volume(mm³) for control (PBS) (⋄) or 10 mg/kg KR-H6-48 treated (□) mice isindicated on the y-axis. The number of days is indicated on the x-axis.

FIG. 10 is a graph illustrating the reduction in activated (GTP-bound)Ras in lung cancer cells by Ras inhibitors. GTP-bound Ras (fraction ofcontrol) for kR-H6-48 (⋄), HR-1 (□), kR-4A-4 (Δ), and kR-48-2 (x) isindicated on the y-axis. The number of hours is indicated on the x-axis.

FIGS. 11A-B are graphs illustrating the growth inhibition ofRas-dependent cancer cells by kR-H6-48 (A) and HR-1 (B). The cell number(percent of control) of H358 (⋄), SKBR3 (□), SKOV3 (Δ), MPR-178 (x), andID8 (•) cells is indicted on the y-axis. The concentration (nM) isindicated on the x-axis.

FIG. 12 illustrates the direct interaction of fluorescent lipopeptideanalogs of Ras. The bound fraction of kR-H6-57 (□), kR-H6-58 (⋄),kR-4B-14 (Δ), HR-6 (∘), and kR-4A-11 (-) in dodecylphosphocholine (DPC)micelles is indicated on the y-axis. The K-Ras-GDP concentration (nM) isindicated on the x-axis. The K_(D) for each inhibitor is as follows:1.44±0.16 μM (kR-H6-57), 0.89±0.13 M (kR-H6-58), 10.8±1.6 μM(kR-4B-14), >100 μM (HR-6), and 11.8±1.3 μM (kR-4A-11).

FIGS. 13A-D illustrate that the affinity of Ras inhibitors towardsrecombinant K-Ras depends on the membrane-mimicking environment and ishigher in bicelles than in micelles. The bound fraction of kR-48-14 inDMPC/DHPC bicelles (A), kR-4B-14 in DPC micelles (B), kR-H6-57 inDMPC/DHPC bicelles (C), and kR-H6-57 in DPC micelles (D) is indicated onthe y-axis. The K-Ras-GDP concentration (nM) is indicated on the x-axis.The K_(D) for each inhibitor is as follows: 1.3±0.1 μM (kR-4B-14 inDMPC/DHPC bicelles), 10.8±1.6 μM (kR-4B-14 in DPC micelles), 86.3±7.6 μM(kR-H6-57 in DMPC/DHPC bicelles), and 1.4±1.6 μM (kR-H6-57 in DPCmicelles).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a peptide or peptidomimetic that is derived fromor based upon the amino acid sequence of the C-terminal α-helix of a Rasprotein, particularly K-Ras-4a, K-Ras-4b, N-Ras, or H-Ras. TheC-terminal α-helix of K-Ras-4a and N-Ras comprises the amino acidsequence of SEQ ID NO: 66. The C-terminal α-helixes of K-Ras-4b andH-Ras comprise the amino acid sequences of SEQ ID NO: 67 and SEQ ID NO:68, respectively. In one aspect, the peptide or peptidomimetic comprisesabout eight or more (e.g., about nine or more, about ten or more, abouteleven or more, or even about 12 or more) contiguous amino acids of theC-terminal α-helix of Ras or inverse thereof, provided that the peptideor peptidomimetic comprises a total of about 30 or fewer amino acids.

In a related aspect, the peptide or peptidomimetic comprises the aminoacid sequence X₁YTLVRX₂X₃RX₄X₅(SEQ ID NO: 5) or the inverse thereof,wherein the peptide or peptidomimetic comprises about 30 or fewer aminoacids. Positions X₁-X₅ of SEQ ID NO: 5 can be any suitable amino acid.Desirably, X₁ is phenylalanine or tryptophan, X₂ is glutamic acid orglutamine, X₃ is isoleucine, valine, or lysine, X₄ is glutamine orlysine, and/or X₅ is tyrosine or histidine. Other amino acids also canbe used, especially those having properties (e.g., size, polar ornon-polar characteristics, charge, and or acid/base properties) similarto the amino acids provided above for any given position. More specificexamples of a peptide or peptidomimetic comprising SEQ ID NO: 5 or theinverse thereof include, but are not limited to, those comprising any ofSEQ ID NOs: 10-46, 66-68, and 75-84.

The invention provides a peptide or peptidomimetic that is derived fromor based upon the amino acid sequence of the hypervariable region (HVR)of a Ras protein, particularly K-Ras-4a, K-Ras-4b, N-Ras, or H-Ras. TheHVRs of K-Ras-4a, K-Ras-4b, H-Ras, and N-Ras comprise the amino acidsequences of SEQ ID NOs: 69-72, respectively. According to one aspect,the peptide or peptidomimetic comprises about five or more (e.g., aboutsix or more, about seven or more, about eight or more, or about nine ormore) contiguous amino acids of the HVR of Ras or inverse thereof,wherein the peptide or peptidomimetic comprises a total of about 30 orfewer amino acids.

In another aspect, the peptide or peptidomimetic comprises the aminoacid sequence KTPGX₁VKIKK (SEQ ID NO: 6) or the inverse thereof, whereinthe peptide or peptidomimetic comprises about 30 or fewer amino acids.X₁ of SEQ ID NO: 6 can be any suitable amino acid. Desirably, X₁ isserine, norleucine, or alanine. Other amino acids also can be used,especially those having similar properties to serine, norleucine, oralanine. More specific examples of a peptide or peptidomimeticcomprising SEQ ID NO: 6 or the inverse thereof include, but are notlimited to, those comprising any of SEQ ID NOs: 47-54 and 69.

Alternatively, the peptide or peptidomimetic comprises the amino acidsequence KKSKTK (SEQ ID NO: 7) or the inverse thereof, wherein thepeptide or peptidomimetic comprises about 30 or fewer amino acids.Particular examples of a peptide or peptidomimetic comprising SEQ ID NO:7 or the inverse thereof include, but are not limited to, thosecomprising any of SEQ ID NOs: 55-63 and 70.

In yet another embodiment, the peptide or peptidomimetic comprises theamino acid sequence SGPGX₁X₂SX₃X₄(SEQ ID NO: 8) or the inverse thereof,wherein the peptide or peptidomimetic comprises about 30 or fewer aminoacids. X₁-X₄ of SEQ ID NO: 8 can be any suitable amino acid. In oneembodiment, X₁-X₃ are non-polar amino acids, and/or X₄ is a non-polar orbasic amino acid. Desirably, X₁ is cysteine or norleucine, X₂ ismethionine or norleucine, X₃ is cysteine or norleucine, and/or X₄ islysine or norleucine. More specific examples of a peptide orpeptidomimetic comprising SEQ ID NO: 8 or the inverse thereof include,but are not limited to, those comprising any of SEQ ID NOs: 64, 71, 85,or 86.

According to still another aspect, the peptide or peptidomimeticcomprises the amino acid sequence GTQGX₁X₂GLP (SEQ ID NO: 9), whereinthe peptide or peptidomimetic comprises about 30 or fewer amino acids.X₁ and X₂ can be any suitable amino acid. According to one embodiment,X₁ and/or X₂ is a non-polar amino acid. In another embodiment, X₁ iscysteine or norleucine, and/or X₂ is methionine or norleucine.Particular examples of a peptide or peptidomimetic comprising SEQ ID NO:9 or the inverse thereof include, but are not limited to, SEQ ID NOs: 65and 72.

Preferably, the peptide or peptidomimetic inhibits Ras activity. Theterm “Ras” is sometimes used herein to refer to the superfamily of Rasproteins collectively and individually, and is intended to encompass anysuch protein, especially H-Ras, N-Ras, K-Ras4A, and K-Ras4B. For thepurposes of this invention, a peptide or peptidomimetic is considered toinhibit Ras activity if it inhibits any biological function of a Rasprotein. Biological functions of Ras proteins include, for example,signal transduction activity. Thus, for instance, a peptide orpeptidomimetic is considered to inhibit Ras activity if, in the presenceof the peptide or peptidomimetic, the signal transduction activity of aRas protein is reduced to any degree as compared to the signaltransduction activity of the Ras protein in the absence of the peptideor peptidomimetic. Preferably, the peptide or peptidomimetic inhibitsRas activity to a degree sufficient to reduce the rate of cell growth ofa cancer cell, reduce malignant transformation of a host, and/or inducecell death of a cancer cell. Suitable assays to test for such areduction in the biological activity of Ras are known in the art,including cell growth and cytotoxicity assays, migration and invasionassays, phosphorylation/de-phosphorylation of Ras downstream targets,and gene regulation assays (e.g., luciferase reporter assay).

The invention is not limited with respect to any particular mechanism ofaction. The peptide or peptidomimetic may inhibit Ras by binding to Rasor its targets, thereby interfering with Ras signal transduction.Alternatively, or in addition, the peptide or peptidomimetic may inhibitthe multimerization of Ras and/or inhibit Ras' membrane bindingactivity. Of course, the peptide or peptidomimetic may act by some othermechanism, such as by increasing the rate of Ras protein degradation orenhancing expression of small RNA molecules (miRNAs) that interfere withRas transcription.

The inventive peptide or peptidomimetic can further comprise one or more(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) flankingresidues. The flanking residues should be chosen so as not to interferewith the ability of the peptide to inhibit Ras activity. Guidance forthe selection of such residues is provided by the relevant sequence ofthe C-terminal α-helix or HVR of Ras itself. For instance, one canchoose residues for use in the peptide that are identical to, or haveproperties similar to, the residues at the corresponding positions of agiven Ras protein (preferably a human Ras protein). The peptide orpeptidomimetic can, for example, be a fragment of a Ras protein or havean amino acid sequence of a fragment of a Ras protein or the inversesequence thereof.

Variant sequences other than those specifically mentioned herein arecontemplated, which comprise significant sequence identity (e.g., 80%,85%, 90%, 95%, 98%, or 99% sequence identity) to the amino acid sequenceof the C-terminal α-helix (e.g., SEQ ID NOs: 66-68) or HVR (e.g., SEQ IDNOs: 69-72) or fragment thereof, provided that such variants retain theability to inhibit Ras activity. Such variants can comprise one or more(e.g., 2, 3, 4, or 5) amino acid substitutions, deletions, or insertionsas compared to the parent amino acid sequence. Conservative amino acidsubstitutions are known in the art, and include amino acid substitutionsin which one amino acid having certain physical and/or chemicalproperties is exchanged for another amino acid that has the same orsimilar chemical or physical properties. For instance, the conservativeamino acid substitution can be an acidic amino acid substituted foranother acidic amino acid (e.g., Asp or Glu), an amino acid with anonpolar side chain substituted for another amino acid with a nonpolarside chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val,etc.), a basic amino acid substituted for another basic amino acid (Lys,Arg, etc.), an amino acid with a polar side chain substituted foranother amino acid with a polar side chain (Asn, Cys, Gin, Ser, Thr,Tyr, etc.), etc.

The peptide or peptidomimetic also can comprise synthetic, non-naturallyoccurring amino acids. Such synthetic amino acids include, for example,aminocyclohexane carboxylic acid, norleucine, α-amino n-decanoic acid,homoserine, S-acetylaminomethyl-cysteine, trans-3- andtrans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine,4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylscrineβ-hydroxyphenylalanine, phenylglycine, α-naphthylalanine,cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid,aminomalonic acid monoamide, N′-benzyl-N′-methyl-lysine,N′,N′-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexane carboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid,α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine,and α-tert-butylglycine. The properties of such synthetic amino acidsare well-documented. Any natural amino acid of one or more of thesequences discussed herein can be substituted with a synthetic aminoacid having similar properties.

The term “peptidomimetic” as used herein refers to a compound thatcomprises the same general structure of a corresponding polypeptide, butwhich includes modifications that increase its stability or biologicalfunction. For instance, the peptidomimetic can be a “retro” or “reverso”analogue of a given peptide, which means that the peptidomimeticcomprises the reverse sequence of the peptide. In addition, or instead,the peptidomimetic can comprise one or more amino acids in a “D”configuration (e.g., D-amino acids), providing an “inverso” analogue.Peptides comprising both a reverse sequence and D-amino acids arereferred to as “retro-inverso” peptides. Peptidomimetics also includepeptoids, wherein the sidechain of each amino acid is appended to thenitrogen atom of the amino acid as opposed to the alpha carbon. Peptoidscan, thus, be considered as N-substituted glycines which have repeatingunits of the general structure of NRCH₂CO and which have the same orsubstantially the same amino acid sequence as the correspondingpolypeptide. In this respect, the peptide or peptidomimetic can compriseany of the sequences described herein in reverse order.

Smaller peptides and peptidomimetics are believed to be advantageous forinhibiting Ras function and to facilitate entry into a cell. Thus, thepeptide or peptidomimetic preferably comprises about 30 or fewer aminoacids, such as about 25 or fewer amino acids, about 20 or fewer aminoacids, or about 15 or fewer amino acids or even about 12 or fewer aminoacids. Generally, however, the peptide or peptidomimetic will compriseabout 8 or more amino acids, such as about 10 or more amino acids, about12 or more amino acids, or about 15 or more amino acids.

The peptide or peptidominietic can comprise, consist essentially of, orconsist of, any of foregoing sequences or variants thereof. The peptideor peptidomimetic consists essentially of the foregoing sequences if itdoes not comprise other elements, such as other amino acid sequences,that prevent the peptide from inhibiting Ras activity.

The peptide or peptidomimetic coupled to a cell penetrating motif orother moiety so as to more efficiently facilitate the delivery of thepeptide to the interior of a cell, anchor the peptide to the cellmembrane of a cell, and/or promote folding of the peptide. Thus, thepeptide or peptidomimetic can be provided as part of a compositioncomprising the peptide and cell penetrating motif or other moiety. Anyof various cell penetrating motifs and or other moieties useful forthese purposes can be used. By way of illustration, suitable cellpenetrating motifs and other relevant moieties (e.g., cell-membraneanchoring moieties) include lipids and fatty acids, peptide transductiondomains (e.g., HIV-TAT, HSV Transcription Factor (VP22), andpenetratin), and other types of carrier molecules (e.g., Pep-1).

According to one aspect of the invention, the cell penetrating motif orother moiety comprises a fatty acid or lipid molecule. The fatty acid orlipid molecule can be, for example, a palmitoyl group, farnesyl group(e.g., farnesyl diphosphate), a geranylgeranyl group (e.g.,geranylgeranyl diphosphate), a phospholipid group,glycophosphatidylinositol, phosphatidylserine, phosphatidylethanolamine,sphingomyelin, phosphatidylcholine, cardiolipin, phosphatidylinositol,phosphatidic acid, lysophosphoglyceride, a cholesterol group, and thelike. Preferably, the fatty acid molecule is a C₁ to C₂₄ fatty acid orC₆ to C₁₄ fatty acid. Desirably, the fatty acid comprises three or more,four or more, five or more, six or more, seven or more, eight or more,nine or more or ten or more carbon atoms. Typically, the fatty acid willcomprise 22 or fewer, 20 or fewer, 18 or fewer, or 16 or fewer carbonatoms. Specific examples of fatty acids include, without limitation,lauric acid, myristic acid, stearic acid, oleic acid, linoleic acid,α-linoleic acid, linolenic acid, arachidonic acid, timnodonic acid,docosohexenoic acid, erucic acid, arachidic acid, behenic acid,aminoisobutiric acid (Aib), caprylic acid (Cap), and octanoic acid(Oct).

The fatty acid or lipid molecule can be attached to any suitable part ofthe peptide or peptidomimetic. In a preferred embodiment of theinvention, the fatty acid or lipid molecule is attached at the amino(N-) terminus, the carboxyl (C-) terminus, or both the N- and C-terminiof the peptide or peptidomimetic. Typically, the fatty acid or lipidmolecule is attached via an amide or ester linkage. When the fatty acidor lipid molecule is attached at the C-terminus of the polypeptide orpeptidomimetic, the fatty acid or lipid molecule preferably is modified,e.g., to include an amino group such as NH₂(CH₂)_(n)COOH orCH₃(CH₂)_(m)CH(NH₂)COOH, wherein each of n and m is, independently, 1 to24, preferably 6 to 14. The fatty acid or lipid residue canadvantageously be attached to a terminal lysine in the epsilon (c)position.

According to another aspect of the invention, the cell penetrating motifis a peptide transduction domain (also known as protein transductiondomains or PTDs). PTDs typically are fused to the Ras-inhibitory peptideor peptidomimetic. Thus, the peptide or peptidomimetic can be a fusionprotein comprising the peptide or peptidomimetic and a PTD. Often, thefusion protein is cleaved inside of a cell to remove the cellpenetrating motif.

The peptide or peptidomimetic can further comprise linking residuesdisposed between the amino acid sequence derived from or based upon theC-terminal α-helix or HVR of Ras and the cell penetrating motif or othermoiety. Illustrative examples of such linking residues include K, KK,RK, RQ, KQ, RQI, KQI, RQIK (SEQ ID NO: 73), and KQIK (SEQ ID NO: 74).

The peptide or peptidomimetic can be prepared by any method, such as bysynthesizing the peptide or peptidomimetic, or by expressing a nucleicacid encoding an appropriate amino acid sequence in a cell andharvesting the peptide from the cell. Of course, a combination of suchmethods also can be used. Methods of de novo synthesizing peptides andpeptidomimetics, and methods of recombinantly producing peptides andpeptidomimetics are known in the art (see, e.g., Chan et al., Fmoc SolidPhase Peptide Synthesis, Oxford University Press, Oxford, UnitedKingdom, 2005; Peptide and Protein Drug Analysis, ed. Reid, R., MarcelDekker, Inc., 2000; Epitope Mapping, ed. Westwood et al., OxfordUniversity Press, Oxford, United Kingdom, 2000; Sambrook et al.,Molecular Cloning: A Laboratory Manual, 3^(rd) ed., Cold Spring HarborPress, Cold Spring Harbor, N. Y. 2001; and Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates and JohnWiley & Sons, NY, 1994).

The invention also provides a nucleic acid encoding the amino acidsequence of the peptide or peptidomimetic. The nucleic acid can compriseDNA or RNA, and can be single or double stranded. Furthermore, thenucleic acid can comprise nucleotide analogues or derivatives (e.g.,inosine or phophorothioate nucleotides and the like). The nucleic acidcan encode the amino acid sequence of the peptide or peptidomimeticalone, or as part of a fusion protein comprising such sequence and acell penetrating motif, as described herein. The nucleic acid encodingthe amino acid sequence of the peptide or peptidomimetic can be providedas part of a construct comprising the nucleic acid and elements thatenable delivery of the nucleic acid to a cell, and/or expression of thenucleic acid in a cell. Such elements include, for example, expressionvectors and transcription and/or translation sequences. Suitablevectors, transcription/translation sequences, and other elements, aswell as methods of preparing such nucleic acids and constructs, areknown in the art (e.g., Sambrook et al., supra; and Ausubel et al.,supra). Accordingly, a recombinant vector comprising the nucleic acidencoding the amino acid sequence of the peptide or peptidomimetic alsois encompassed by the invention.

The invention further provides an antibody to the peptide orpeptidomimetic, or an antigen binding fragment or portion thereof (e.g.,Fab, F(ab′)₂, dsFv, sFv, diabodies, and triabodies). The antibody can bemonoclonal or polyclonal, and of any isotype, e.g., IgA, IgD, IgE, IgG,IgM, etc. The antibody can be a naturally-occurring antibody, e.g., anantibody isolated and/or purified from a mammal, e.g., mouse, rabbit,goat, horse, chicken, hamster, human, etc. Alternatively, the antibodycan be a synthetic or genetically-engineered antibody, e.g., a humanizedantibody or a chimeric antibody. The antibody can be in monomeric orpolymeric form. The antibody, or antigen binding portion thereof, can bemodified to comprise a detectable label, such as, for instance, aradioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC),phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradishperoxidase), or element particles (e.g., gold particles). Suchantibodies can be used for any purpose, such as to facilitate thedetection or purification of a peptide or peptidomimetic describedherein. Suitable methods of making antibodies are known in the art,including standard hybridoma methods, EBV-hybridoma methods,bacteriophage vector expression systems, and phage-display systems (see,e.g., Köhler and Milstein, Eur. J. Immunol., 5, 511-519 (1976); Harlowand Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988); C.A. Janeway et al. (eds.), Immunobiology, 5^(th) Ed., Garland Publishing,New York, N.Y. (2001); Haskard and Archer, J. Immunol. Methods, 74(2),361-67 (1984); Roder et al., Methods Enzymol., 121, 140-67 (1986); Huseet al., Science, 246, 1275-81 (1989); Sambrook et al., supra; Ausubel etal., supra; and Knappik et al., J. Mol. Biol. 296: 57-86 (2000)).

The peptide or peptidomimetic, nucleic acid, or antibody can beisolated. The term “isolated” as used herein encompasses compounds orcompositions that have been removed from a biological environment (e.g.,a cell, tissue, culture medium, body fluid, etc.), or otherwiseincreased in purity to any degree (e.g., isolated from a synthesismedium). Isolated compounds and compositions, thus, can be synthetic ornaturally produced.

A cell comprising the peptide or peptidomimetic, nucleic acid encodingthe amino acid sequence of the peptide or peptidomimetic, or recombinantvector comprising the nucleic acid also is provided herein. Such a cellincludes, for example, a cell engineered to express a nucleic acidencoding the amino acid sequence of the peptide or peptidomimetic.Suitable cells include prokaryotic and eukaryotic cells, e.g., mammaliancells, yeast, fungi, and bacteria (such as E. coli). The cell can be invitro, as is useful for research or for production of the peptide orpeptidomimetic, or the cell can be in vivo, for example, in a transgenicmammal that expresses the peptide.

The peptide or peptidomimetic can be used for any purpose, but isespecially useful for inhibiting Ras activity in a cell. Thus, providedherein is a method of inhibiting Ras activity in a cell, which methodcomprises administering a peptide or peptidomimetic described herein toa cell in an amount sufficient to inhibit Ras activity.

The peptide or peptidomimetic can be administered to the cell by anymethod. For example, the peptide or peptidomimetic can be administeredto a cell by contacting the cell with the peptide or peptidomimetic,typically in conjunction with a reagent or other technique (e.g.,microinjection or electroporation) that facilitates cellular uptake.Alternatively, and preferably, the peptide or peptidomimetic isadministered by contacting the cell with a composition comprising thepeptide or peptidomimetic and a cell penetrating motif, as discussedherein. The peptide or peptidomimetic additionally or alternatively canbe encapsulated in nanoparticles (e.g., using the methods described inInternational Patent Application Publication 2008/058125) prior toadministration to the cell.

The peptide or peptidomimetic also can be administered by introducing anucleic acid encoding the amino acid sequence of the peptide into thecell such that the cell expresses a peptide comprising the amino acidsequence. The nucleic acid encoding the peptide can be introduced intothe cell by any of various techniques, such as by contacting the cellwith the nucleic acid or a composition comprising the nucleic acid aspart of a construct, as described herein, that enables the delivery andexpression of the nucleic acid. Specific protocols for introducing andexpressing nucleic acids in cells are known in the art (see, e.g.,Sambrook et al. (eds.), supra; and Ausubel et al., supra).

The peptide, peptidomimetic, or nucleic acid can be administered to acell in vivo by administering the peptide, peptidomimetic, nucleic acidencoding the peptide or peptidomimetic, or recombinant vector comprisingthe nucleic acid. The host can be any host, such as a mammal, preferablya human. Suitable methods of administering peptides, peptidomimetics,and nucleic acids to hosts are known in the art, and discussed ingreater detail in connection with the pharmaceutical compositioncomprising such compounds, below.

The cell can be any type of cell that comprises Ras. Preferably, thecell is of a type that is related to a disease or condition mediated byRas activity. For example, the cell can be an engineered cell that isdesigned to mimic a condition or disease associated with Ras activity,or the cell can be a cell of a patient afflicted with a disease orcondition associated with Ras activity. Diseases mediated by Ras includediseases characterized by Ras overexpression or overactivity. Cancercells are one example of a cell type that can be used. The cell can bein vitro or in vivo in any type of animal, such as a mammal, preferablya human.

The method of inhibiting Ras activity in a cell can be used for anypurpose, such as for the research, treatment, or prevention of diseasesor conditions mediated by Ras. Ras activity has been linked to a largevariety of cancers. Thus, according to one aspect of the method of theinvention, the peptide or peptidomimetic is administered to a cancercell, in vitro or in vivo, and administration of the peptide orpeptidomimetic to the cancer cell inhibits the growth or survival of thecancer cell.

The cancer cell can be a cell of any type of cancer, in vitro or invivo, particularly those associated with Ras activity, such as thoseassociated with Ras overexpression, up-regulation of Ras, and/orincreased activation of Ras (e.g., constitutive activation of Ras).Non-limiting examples of specific types of cancers include cancer of thehead and neck, eye, skin, mouth, throat, esophagus, chest, bone, lung,colon, sigmoid, rectum, stomach, prostate, breast, ovaries, kidney,liver, pancreas, brain, intestine, heart or adrenals. More particularly,cancers include solid tumor, sarcoma, carcinomas, fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, Kaposi's sarcoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma, retinoblastoma, a blood-born tumor, acutelymphoblastic leukemia, acute lymphoblastic B-cell leukemia, acutelymphoblastic T-cell leukemia, acute myeloblastic leukemia, acutepromyelocytic leukemia, acute monoblastic leukemia, acuteerythroleukemic leukemia, acute megakaryoblastic leukemia, acutemyelomonocytic leukemia, acutenonlymphocyctic leukemia, acuteundifferentiated leukemia, chronic myelocytic leukemia, chroniclymphocytic leukemia, hairy cell leukemia, or multiple myeloma. See,e.g., Harrison's Principles of Internal Medicine, Eugene Braunwald etal., eds., pp. 491 762 (15th ed. 2001). The methods of the invention arebelieved to be especially useful for the treatment of leukemia,pancreatic cancer, colon (colorectal) cancer, ovarian cancer, lungcancer, bladder cancer, cancer of the salivary gland, myeloidmalignancies, germ cell tumors, and melanoma, as well as any othercancer known to be responsive to Ras inhibitors.

Ras activity also has been linked to other diseases, including Costellosyndrome, Noonan syndrome, and autoimmune diseases, such as Alopeciaareata, Ankylosing spondylitis, Crohns Disease, Graves' disease,Dermatomyositis, Diabetes mellitus type 1, Goodpasture's syndrome,Guillain-Barré syndrome (GBS), Hashimoto's disease, Idiopathicthrombocytopenic purpura, Lupus erythematosus, Mixed Connective TissueDisease, Multiple Sclerosis, Myasthenia gravis, Narcolepsy, Pemphigusvulgaris, Pernicious anaemia, Psoriasis, Psoriatic Arthritis,Polymyositis, Primary biliary cirrhosis, Relapsing polychondritis,Rheumatoid arthritis, Sjögren's syndrome, Temporal arteritis (also knownas “giant cell arteritis”), Ulcerative Colitis (one of two types ofidiopathic inflammatory bowel disease “IBD”), Vasculitis, and Wegener'sgranulomatosis. Thus, the methods of the invention are believed to beuseful for the treatment of such diseases, as well.

Peptides and peptidomimetics, as described herein, include salts,esters, alkylated (e.g., methylated), and acetylated peptides. Any oneor more of the compounds or compositions of the invention describedherein (e.g., peptide or peptidomimetic, nucleic acid, antibody, orcell) can be formulated as a pharmaceutical composition, comprising acompound of the invention and a pharmaceutically acceptable carrier.Furthermore, the compounds or compositions of the invention can be usedin the methods described herein alone or as part of a pharmaceuticalformulation.

The pharmaceutical composition can comprise more than one compound orcomposition of the invention. Alternatively, or in addition, thepharmaceutical composition can comprise one or more otherpharmaceutically active agents or drugs. Examples of such otherpharmaceutically active agents or drugs that may be suitable for use inthe pharmaceutical composition include anticancer agents. Suitableanticancer agents include, without limitation, alkylating agents;nitrogen mustards; folate antagonists; purine antagonists; pyrimidineantagoinists; spindle poisons; topoisomerase inhibitors; apoptosisinducing agents; angiogenesis inhibitors; podophyllotoxins;nitrosoureas; cisplatin; carboplatin; interferon; asparginase;tamoxifen; leuprolide; flutamide; megestrol; mitomycin; bleomycin;doxorubicin; irinotecan; and taxol, geldanamycin (e.g., 17-AAG), andvarious anti-cancer peptides and antibodies.

The carrier can be any of those conventionally used and is limited onlyby physio-chemical considerations, such as solubility and lack ofreactivity with the active compound(s), and by the route ofadministration. The pharmaceutically acceptable carriers describedherein, for example, vehicles, adjuvants, excipients, and diluents, arewell-known to those skilled in the art and are readily available to thepublic. It is preferred that the pharmaceutically acceptable carrier beone which is chemically inert to the active agent(s) and one which hasno detrimental side effects or toxicity under the conditions of use.

The choice of carrier will be determined in part by the particularcompound or composition of the invention and other active agents ordrugs used, as well as by the particular method used to administer thecompound and/or inhibitor. Accordingly, there are a variety of suitableformulations of the pharmaceutical composition of the present inventivemethods. The following formulations for oral, aerosol, parenteral,subcutaneous, intravenous, intramuscular, interperitoneal, rectal, andvaginal administration are exemplary and are in no way limiting. Oneskilled in the art will appreciate that these routes of administeringthe compound of the invention are known, and, although more than oneroute can be used to administer a particular compound, a particularroute can provide a more immediate and more effective response thananother route.

Injectable formulations are among those formulations that are preferredin accordance with the present invention. The requirements for effectivepharmaceutical carriers for injectable compositions are well-known tothose of ordinary skill in the art (See, e.g., Pharmaceutics andPharmacy Practice, J. B. Lippincott Company, Philadelphia, Pa., Bankerand Chalmers, eds., pages 238-250 (1982), and ASHP Handbook onInjectable Drugs. Toissel, 4th ed., pages 622-630 (1986)).

Topical formulations are well-known to those of skill in the art. Suchformulations are particularly suitable in the context of the presentinvention for application to the skin.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the inhibitor dissolved indiluents, such as water, saline, or orange juice; (b) capsules, sachets,tablets, lozenges, and troches, each containing a predetermined amountof the active ingredient, as solids or granules; (c) powders; (d)suspensions in an appropriate liquid; and (e) suitable emulsions. Liquidformulations may include diluents, such as water and alcohols, forexample, ethanol, benzyl alcohol, and the polyethylene alcohols, eitherwith or without the addition of a pharmaceutically acceptablesurfactant. Capsule forms can be of the ordinary hard- or soft-shelledgelatin type containing, for example, surfactants, lubricants, and inertfillers, such as lactose, sucrose, calcium phosphate, and corn starch.Tablet forms can include one or more of lactose, sucrose, mannitol, cornstarch, potato starch, alginic acid, microcrystalline cellulose, acacia,gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium,talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid,and other excipients, colorants, diluents, buffering agents,disintegrating agents, moistening agents, preservatives, flavoringagents, and pharmacologically compatible excipients. Lozenge forms cancomprise the active ingredient in a flavor, usually sucrose and acaciaor tragacanth, as well as pastilles comprising the active ingredient inan inert base, such as gelatin and glycerin, or sucrose and acacia,emulsions, gels, and the like containing, in addition to the activeingredient, such excipients as are known in the art.

The compounds and compositions of the invention, alone or in combinationwith other suitable components, can be made into aerosol formulations tobe administered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like. They also maybe formulated as pharmaceuticals for non-pressured preparations, such asin a nebulizer or an atomizer. Such spray formulations also may be usedto spray mucosa.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The compounds and compositions of the invention can be administered in aphysiologically acceptable diluent in a pharmaceutical carrier, such asa sterile liquid or mixture of liquids, including water, saline, aqueousdextrose and related sugar solutions, an alcohol, such as ethanol,isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol orpolyethylene glycol, dimethylsulfoxide, glycerol ketals, such as2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, such aspoly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester orglyceride, or an acetylated fatty acid glyceride with or without theaddition of a pharmaceutically acceptable surfactant, such as a soap ora detergent, suspending agent, such as pectin, carbomers,methylcellulose, hydroxypropylmethylcellulose, orcarboxymethylcellulose, or emulsifying agents and other pharmaceuticaladjuvants.

Oils, which can be used in parenteral formulations include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters.

Suitable soaps for use in parenteral formulations include fatty alkalimetal, ammonium, and triethanolamine salts, and suitable detergentsinclude (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides, (b) anionic detergentssuch as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionicdetergents such as, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylenepolypropylene copolymers, (d)amphoteric detergents such as, for example, alkyl-b-aminopropionates,and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixturesthereof.

Preservatives and buffers may be used. In order to minimize or eliminateirritation at the site of injection, such compositions may contain oneor more nonionic surfactants having a hydrophile-lipophile balance (HLB)of from about 12 to about 17. The quantity of surfactant in suchformulations will typically range from about 5% to about 15% by weight.Suitable surfactants include polyethylene sorbitan fatty acid esters,such as sorbitan monooleate and the high molecular weight adducts ofethylene oxide with a hydrophobic base, formed by the condensation ofpropylene oxide with propylene glycol. The parenteral formulations canbe presented in unit-dose or multi-dose sealed containers, such asampoules and vials, and can be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid excipient,for example, water, for injections, immediately prior to use.Extemporaneous injection solutions and suspensions can be prepared fromsterile powders, granules, and tablets of the kind previously described.

Additionally, the compounds of the invention, or compositions comprisingsuch compounds, can be made into suppositories by mixing with a varietyof bases, such as emulsifying bases or water-soluble bases. Formulationssuitable for vaginal administration can be presented as pessaries,tampons, creams, gels, pastes, foams, or spray formulas containing, inaddition to the active ingredient, such carriers as are known in the artto be appropriate.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLES

Except otherwise stated, the peptides referenced in the followingexamples were prepared and analyzed as follows:

Peptide Synthesis and Purification

The peptides were synthesized on a 433A Peptide Synthesizer (AppliedBiosystems) using Fmoc chemistry. The peptides were cleaved from theresin and deprotected with a mixture of 90.0% (v/v) trifluoroacetic acid(TFA) with 2.5% water, 2.5% triisopmrpyl-silane, and 5% thioanisol. Theresin and deprotection mixture were pre-chilled to −5° C. and reactedfor 15 minutes at −5° C. with stirring. The reaction was allowed tocontinue at room temperature for 1 hour and 45 minutes. The resin wasfiltered off and the product was precipitated with cold diethyl ether.The resin was washed with neat TFA. Peptide suspended in diethyl etherwas centrifuged at −20° C. and the precipitate was washed with diethylether four more times and left to dry in a vacuum overnight. The driedcrude peptide was dissolved in DMSO and purified on a preparative (25mm×250 mm) Atlantis C18 reverse phase column (Agilent Technologies) in a90 minute gradient of 0.1% (v/v) trifluoroacetic acid in water and 0.1%trifluoroacetic acid in acetonitrile with a 10 mL/min flow rate. Thefractions containing peptides were analyzed on Agilent 1100 LC/MSspectrometer with the use of a Zorbax 300SB-C3 Poroshell column and agradient of 5% acetic acid in water and acetonitrile. Fractions thatwere more than 95% pure were combined and freeze dried. Resin preloadedwith α-Fmoc-ε-palmytoil-Lys was prepared as described in Remsberg etal., J. Med. Chem., 50: 4534-4538 (2007).

Cell Toxicity Assay

MCF-7 (breast cancer), T47D (breast cancer), Cole 205 (colon cancer),JM-1 (rat hepatoma), Sk Mel-2 (melanoma), PLC (human hepatoma), andHepG2 (human hepatoma) were obtained from American Type Cell CultureCollection. MCF-7 cells were grown in RPMI medium supplemented with 10%Fetal Bovine Serum. The remaining cell lines were grown in DMEM mediumsupplemented with 10% Fetal Bovine Serum. For the assay, cells wereseeded into 96 well plates in medium containing 1% Fetal Bovine Serumand 100 μL of a cell suspension containing 5000 cells per well were usedfor each well. Cell growth was evaluated utilizing MTT((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium). The absorbanceof the wells at 544 nm was determined by a FLUOstar/POLARstar Galaxy(BMG Lab Technologies GmbH) microplate reader.

Cell Invasion Assays

Cell invasion assays were performed using the BD Biocoat MatrigelInvasion Chambers in accordance with the manufacturer's protocol. Cells(1×10⁵/mL) were seeded onto 12-well cell culture chamber using insertswith 8 μm pore size polycarbonate membrane over a thin layer of MatrigelBasement Membrane Matrix without phenol red (BD Biosciences) diluted at1:100 in PBS. Following incubation of the plates for 48 hours at 37° C.,cells that invaded through the Matrigel and migrated to the lowersurface of the membrane were stained with Giemsa solution, counted underthe microscope in at least 10 different fields, and photographed. Threewells were examined for each condition and cell type, and theexperiments were repeated in triplicate.

Example 1

This example demonstrates the identification of analogs of theC-terminal α-helix of Ras.

The design of inhibitors was based on available x-ray structures ofK-Ras and H-Ras proteins. The structures have suggested that theC-terminal α-helix of the proteins (helix 6 in K-Ras) occupies centralposition in the protein, is involved in multiple intramolecularinteractions, and, thus, is likely to play a critical role in bothstructure stabilization and structural rearrangements during signalingevents.

A library of synthetic peptide analogs of helix 6 was constructed. Forstructural stabilization of protein fragments and membrane anchoring,all peptide analogs were equipped with palmitate residues (see Table 1).The sequences of the peptides are presented in Table 1, wherein aminoacid substitutions in the native sequences are in bold.

TABLE 1 Structure-activity relationships in derivatives of theC-terminal α-helix of K-Ras. SEQ ID Compound NO Structure GI₅₀, nMkR-H6-1 10    Pal-RYQRIERVLTYFADEV (All D)  1350 kR-H6-2 11  Pal-LRYQRIERVLTYFADEV (All D)   300 kR-H6-3 12e-Pal-KRYQRIERVLTYFADEV (All D) >5000 kR-H6-4 13    Pal-KVEDAFYTLVREIRQYR >5000 kR-H6-5 14   Ac-Doa-VEDAFYTLVREIRQYR 3300 kR-H6-6 15      Pal-e-KAFTYLVREIRQYR   250 kR-H6-7 16      Ac-VEDAFYTLVREIRQYRK (e-Pal)  2700 kR-H6-8 17         Ac-AFYTLVREIRQYRK (e-Pal)  5000 kR-H6-9 18        Ac-DAFYTLVREIRQYRK (e-Pal)   500 kR-H6-10 19     Pal-e-KAFYTLVREIRQY   300 kR-H6-12 20       Pal-e-DFYTLVREIRQYR  180 kR-H6-11 21      Ac-YQRIQRVLTYFK-e-Pal (all D)    10 kR-H6-13 22     Ac-YQRKQRVLTYFK-e-Pal (all D)    60 kR-H6-14 23     Pal-YQRKQRVLTYF       (all D)    40 kR-H6-16 24         Ac-VEDAFYTLVREIRQYR >5000 kR-H6-17 25            Ac-AFYTLVREIRQYR >5000 kR-H6-18 26        Pal-e-KAFYTLVREIRKHK >1000 kR-H6-19 27     Pal-RYQRIQRVLTYFA (all D) >1000 kR-H6-20 28     Pal-RYQRIQRVLTYF (all D)   250 kR-H6-21 29      Pal-YQRIQRBLTYFA (all D)    30 kR-H6-22 30     Pal-RYQRVQRVLTYFA (all D) In testing kR-H6-23 31      Ac-RYQRIEFVLTYFAK-e-Pal (all D) 25 ± 10 kR-H6-27 32         Pal-e-KAFYTLVREIRQYRL 280 ± 30  kR-H6-23 33         Pal-e-KAFYTLVRQIRQYRL 250 ± 50  kR-H6-30 34      Ac-RYQRIQRVLTYFAK-e-Pal (all D) 10 ± 5  kR-H6-31 35     Ac-LRYQRIQRVLTYFAK-e-Pal (all D) 25 ± 10 kR-H6-32 36      Ac-RYQRIQRVLTYFK-e-Pal (all D) 10 ± 4  kR-H6-33 37       Ac-YQRIQRVLTYFAK-e-Pal (all D) 4.5 ± 1   kR-H6-34 38      Pal-YQRIQRVLTYF (all D) 18 ± 5  kR-H6-35 39  Pal-Aib-YQRIQRVLTYF (all D) 450 ± 50  kR-H6-36 40      Pal-YQRVQRVLTYF (all D) 10 ± 5  kR-H6-38 41      Lau-YQRVQRVLTYF (all D)    15 kR-H6-39 42      Lau-YQRVQRVLTYW (all D) 90 ± 15 kR-H6-40 43      Myr-YQRVQRVLTYW (all D) 20 ± 10 kR-H6-41 44      Cap-YQRVQRVLTYW (all D) 180 ± 40  kR-H6-42 45      Lau-YKRVQRVLTYF (all D) 300 ± 50  kR-H6-46 46      Lau-HQRVQRVLTYF (all D) 300 ± 50  kR-H6-75 75      Lau-WQRVQRVLTYF (all D) 1 ± 1 kR-H6-76 76      Lau-YQRVQRVLTYFC (all D) In testing kR-H6-77 77       Ac-YQRVQRVLTYFC (all D) In testing kR-H6-78 78      Lau-YQRVQRVLTYFA (all D) 1 ± 1 kR-H6-79 79      Cap-YQRVQRVLTYFA (all D) 0.8 ± 0.5 kR-H6-80 80      Cap-YQRVQRVLTYF (all D)        0.8 kR-H6-81 81      Oct-YQRVQRVLTYF (all D) 4 ± 1 kR-H6-82 82      Lau-YQRVQRVLTYFC(Fluo) (all D) In testing kR-H6-83 83      Oct-YQRVQRVLTYFA (all D) In testing kR-H6-84 84      Oct-WQRVQRVLTYFA (all D) In testing Ac = acetylatation Aib =amino-isobutiric acid Doa = 2-dodecyl-alanine Pal = palmtic acid Urn =lauric or dodecanoic acid Cap = caprylic or decanoic acid Oct = octanoicacid

The growth inhibitory activity of compounds was compared using the A549human lung cancer cell line harboring constitutively active G12D Rasmutant with the help of an MTT assay. Peptide kR-H6-12 was the mosteffective in inhibiting cell growth with GI₅₀=180 nM (see Table 1).Stepwise extensions and truncations confirmed that the peptide had theoptimal length (see Table 1).

Longer peptides (e.g., kR-H6-6) had lower potency. Further extensionsled to further reduction in activity, possibly due to incorporation ofnegatively charged residues that interfere with cell entry (e.g.,kR-H6-4). Compounds with palmitate on the N-terminal end weresignificantly more active than compounds with palmitic acid on theC-terminus (kR-H6-6 compared to kR-H6-8). Retro-inverso versions of thepeptide turned out to be significantly more potent. For example, theretroinverso version of kR-H6-6 (kR-H6-23) was 10-fold more toxic tocancer cells (see Table 1).

Analysis of Ras structures suggested that Glu162 is not involved information of any salt bridges. Since negative charges interfere withcell entry, Glu162 was replaced with uncharged Gln, which resulted inadditional 1.7-fold reduction in GI₅₀ (kR-H6-30). Incorporation ofaminoisobutiric acid (Aib) is known to stabilize the helical fold ofpeptides and, therefore, was incorporated on the termini of the helix 6derivatives to improve efficacy of inhibitors by facilitating moreefficient folding and thus better mimicking the parent structure.

Example 2

This example demonstrates the characterization of the interaction ofanalogs of the C-terminal α-helix with Ras protein.

To study the interactions of the catalytic domain of K-Ras with kR-H6-6,¹⁵N-labeled truncated K-Ras (1-166) lacking the hypervariable region wasprepared as described in (Abraham et al., Protein Expression andPurification, 73(2): 125-131 (2010)). The purified protein wasconcentrated to 200 μM and titrated with a solution of kR-H6-6. Thetitration was followed by NMR ¹⁵N-edited HSQC spectra acquired on a 600MHz Bruker spectrometer at 25° C.

Analysis of NMR titration data revealed a localized interaction of thepeptide with the Switch I region of K-Ras with the most significantchemical shift perturbations found in residues Q25, N26, S32, T35, andE37. Intermolecular interactions between helix 6 of GTP-γ-S loaded K-Rasand the Switch I region of the neighboring K-Ras molecule previouslywere observed by x-ray crystallography. The Switch I region of Ras iscritical for nucleotide binding and hydrolysis, and for interactionswith effector proteins. Therefore, it is not surprising that kR-H6-6binding to the Switch I region may interfere with K-Ras signaling.

Example 3

This example demonstrates the identification of analogs of thehypervariable region (HVR) of Ras.

The HVR of Ras protein has been suggested to be involved in targetingproteins to certain regions of cellular membranes and in interactionswith effector proteins. HVR of Ras protein is naturally lipidated on theC-terminal ends. To generate the mimetics of HVR, peptides palmytilatedon the C-terminal ends through ε-amino group of Lys that replacedfarnesylated Cys of Ras were synthesized. The sequences of the peptidesare presented in Table 2.

Since there was no structural data for any HVR of Ras, extensivestructure-activity studies along with comparison of equivalent sequencesin all isoforms of human Ras had to be undertaken (see FIG. 1 and Table2). K-Ras-4A and N-Ras were palmitoylated on Cys 180 and 181,respectively, and were farnesylated on Cys 186, while H-Ras waspalmitoylated on Cys 181 and 184 and farnesylated on Cys 186. Formimicking the palmitoylated Cys 180, 181, and 184 without significantdecrease in peptide solubility, structurally similar, but lesshydrophopic, norleucine residues (L_(N)) were introduced in the sequenceof peptides.

The growth inhibitory activity of compounds was compared using the A549human lung cancer cell line harboring constitutively active G12D Rasmutant with the help of an MIT assay. Optimization of compounds resultedin peptides that were significantly less potent than derivatives ofhelix 6; however, the compounds showed promising activity in

TABLE 2 Structure-activity relationships in derivatives of thehypervariable regions of Ras. SEQ ID GI₅₀, Compound NO Structure μM kR-4A-1 47        Ac-RLKKISKEEKTPGSVKIKKK-e-Pal 3.1 kR-4A-2 48      Ac-YRLKKISKEEKTPGK-e-Pal 1.65 kR-4A-4 49                Ac-KTPGL_(N)VKIKKK-e-Pal 0.7 kR-4A-3 50      Ac-YRLKKISKEEKTPGL_(N)VKIKKK-e-Pal 1.9 kR-4A-5 51                Ac-KTPGL_(N)VKIKKK-e-Pal 5 kR-4A-6 52                 Ac-TPGL_(N)VKIKKK-e-Pal >10 kR-4A-7 53                  Ac-PGL_(N)VKIKKK-e-Pal >10 kR-4A-8 54                   Ac-GL_(N)VKIKKK-e-Pal >10 kR-4B-1 55Ac-KEKL_(N)SKDGKKKKKKSKTKK-e-Pal 1.45 kR-4B-2 56  Ac-KL_(N)SKDGKKKKKKSKTKK-e-Pal 1.4 kR-4B-3 57         Ac-KKKKKKSKTKK-e-Pal 1.75 kR-4B-4 58         Ac-KKKKKKSKTK-e-Pal 1.45  (0.5) kR-4B-6 59   Pal-KTKSKKKKK-NH₂ All-D 1.7 kR-4B-9 60 e-Pal-KKTKSKKKKK-NH₂ All-D 1.4kR-4B-7 61          Ac-KKKKSKTKK-e-Pal 2 kR-4B-8 62          Ac-KKKSKTKK-e-Pal 2.3 kR-4B-10 63            Ac-KKSKTKK-e-PalIn testing HR-1 64          Ac-ESGPGL_(N)L_(N)SL_(N)KK-e-Pal 0.25 HR-285         Pal-KL_(N)L_(N)SL_(N)GPGSE-NH₂ In testing HHR-3 86        Lau-KL_(N)L_(N)SL_(N)GPGSE-NH₂ In testing NR-1 65         Ac-DGTQGL_(N)L_(N)GLPK-e-Pal >10

Example 4

This example demonstrates the membrane binding activity of analogs ofthe HVR of Ras.

Membrane binding is thought to be important for Ras transformingactivity. To assess the ability of K-Ras inhibitors to accumulate on themembrane, stabilized phospholipid bilayer nanodiscs were used as plasmamembrane mimics.

Dipalmitoylphosphatidylcholine (DPPC) bilayers containing 5%dipalmitoyldiphosphatidylethanolamine (DPPE) stabilized by MSP1 scaffoldprotein were prepared. The DPPC lipids were chosen because they areamong the most common components of the plasma membrane. The purifiedMSP1 protein was a generous gift from Dr. Sligar's lab, University ofIllinois at Urbana, which also provided the procedure for preparation ofnanodiscs. The preparation procedure involved mixing DPPC lipids withMSP1 at a 90:1 molar ratio in the presence of 100 mM cholate. Cholatewas removed by slow dialysis. The assembled nanodiscs were purified bysize-exclusion chromatography on a calibrated Superdex 200 column andimmobilized on a surface plasmon resonance (SPR) sensor chip.

The presence of a primary amine group in DPPE provided a convenient sitefor cross-linking of nanodiscs to an SPR sensor chip. Thenoncross-linked dextran in the sample and reference cells was blocked bythe coupling reagent. Four different peptides were used to studymembrane binding by SPR. The HVR peptide represents the hypervariableregion of K-Ras-4B and contains residues 165 through 183. The HVRpeptide lacks post-translational modifications present in K-Ras4B. Thesecond peptide is the HVR peptide conjugated to S-farnesyl L-cysteinemethyl ester via a bifunctional cross-linkerSulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(Sulfo-SMCC) (Pierce). The other two peptides were kR-4A-C1 andkR-4B-C1.

In all cases, with the exception of the HVR peptide, cooperativeinteraction with the phospholipid bilayers was observed. In thissituation determination of true dissociation constants was not feasible.Instead, the data were analyzed using the Hill equation. The Hillcoefficient describes the degree of binding cooperativity. Whilecooperativity for the HVR peptide was low with the Hill coefficient of1.106±0.001, lipid modification significantly increased membrane bindingcooperativity with the highest Hill coefficient of 6.15±0.02 observedfor kR-4B-C1. The Hill coefficient for the HVR peptide modified withS-farnesyl L-cysteine methyl ester was 5.725±0.002. Among lipidatedpeptides kR-4A-C1 exhibited the lowest membrane binding cooperativitywith the Hill coefficient of 3.606±0.003.

Cooperative interaction of the peptides with the membrane phospholipidsnot only enhances their binding affinity but also facilitates theirclustering on the plasma membrane for a more potent effect on K-Ras.Among the peptides studied for their binding to the phospholipidbilayers, the palmitoylated kR-4B-1 peptide showed the best cooperativeinteractions with membrane lipids. The ability of kR-4B-1 to cluster onthe phospholipid bilayers was superior even to the HVR with nativefarnesyl and methyl ester modifications.

Example 5

This example demonstrates the biological activity of Ras inhibitors.

Activity of compounds towards different cell lines varied significantlyeven if the cells expressed identical versions of mutated Ras. Relativeactivities for different cells were similar for compounds mimickinghelix 6 and HVR of K-Ras (see FIGS. 2 and 3), suggesting that they mayact by similar mechanisms in spite of targeting different regions of Rasprotein. Interestingly, mouse cell line 187436 tu2 with well-definedgenetic alterations was the most sensitive to the compounds (see FIGS. 2and 3). The cell line was generated from astrocytoma developed in atransgenic mouse harboring a G12D mutation in K-Ras and inactivation ofthe Rb gene.

Human lung cancer cell lines, A549, H460, and HI 1944, are known to haveadditional oncogenic mutations. Consequently, these cell lines arelikely to have populations of cells resistant to Ras inhibitors and,thus, will appear less sensitive to the compounds. Human lung cancercell lines with identical Ras mutations also have been reported to havedifferent degree of “Ras addiction” or sensitivity to Ras inhibition.

The inventive Ras inhibitors were much more toxic even to less sensitivelung cancer cells than to regular immortalized fibroblasts (see FIG. 4).No killing of epithelial cells was observed even for micromolarconcentrations of the inhibitors suggesting that a therapeutic windowshould exist that will allow selective elimination of tumor cells withthe help of the inventive helix 6 analogs.

Analogs of HVR were on average twice as potent in growth inhibition ofcells with mutated K-Ras as the cells with wild-type protein (see Table3).

TABLE 3 Growth inhibitor activity of HVR analogs evaluated on thoracicmalignancy cell lines. Cell K-Ras kR-4A-3 kR-4B-3 Tumor type line statusGI₅₀ (μM) GI₅₀ (μM) lung adenocarcinoma A549 Mutant 3.1 1.45 lungadenocarcinoma H2009 Mutant 2.2 2.9 lung adenocarcinoma H23 Mutant 2.62.4 lung adenocarcinoma Calu 6 Mutant 3.0 2.7 broncheoalveolar H358Mutant 2.2 2.0 lung squamous cell carcinoma H157 Mutant 2.4 1.5 largecell carcinoma H460 Mutant 1.5 1.0 lung adenocarcinoma Calu3 WT 5.9 2.2lung adenocarcinoma H2882 WT 4.0 3.0 lung squamous cell carcinoma HCC95WT 5.1 3.3 lung squamous cell carcinoma HCC15 WT 7.4 3.1 MPM H2373 WT6.1 4.8 MPM H2461 WT >10.0 >10.0 MPM H2596 WT 8.2 6.2 MPM HP-1 WT >107.3 MPM = malignant pleural mesothelioma WT = wild-type

Although not wishing to be bound by any theory, the inventorshypothesize that the differences in sensitivity are relatively smallbecause Ras gets activated in tumor cells by multiple mechanismsincluding activation of receptor tyrosine kinases. Since Ras functionsdownstream of important regulators of tumor cell growth, such asepithelial growth factor receptor, insulin-like growth factor 1receptor, and MET, its activation is a common event in tumors. Thus,wide-spread sensitivity of tumor cell lines to Ras inhibitors is notsurprising.

Using a “wound closure” assay on HCC15 cells, wherein the closure of a“wound” or gap in a cell monolayer is monitored and measured,demonstrated remarkable reduction in cell motility in the presence ofanalogs of HVR and helix 6 (see FIG. 5). The rate of migration andinvasion was determined using Boyden Chambers (Millipore) in accordancewith the manufacturer's protocol. The migration and invasion rate wasdramatically reduced in the presence of IC₂₅ concentration of theanalogs (see FIGS. 6 and 7). The data suggests that Ras inhibitors havean effect on tumor growth and its metastatic ability.

A further experiment was undertaken to determine the effect of analogsof helix 6 and HVR on the amount of K-Ras protein in cancer cells. H2009(lung adenocarcinoma cells) and H2592 (plural mesothelioma cells) wereexposed to varying concentrations (0-3.0 μM) of kR-4A-3, kR-4B-3, orkR-H6-3 for 18 hours. The cells then were lysed and the lysate wasanalyzed by Western blot using anti-Ras mAb (Cell Biolabs, Inc.).Inhibitors of Ras reduced the amount of Ras protein in lung cancer cellsin a concentration-dependent manner.

H2592 cells were exposed to GI₅₀ concentrations of kR-4A-3 or kR-H6-3for 18 hours, fixed with 4-7% (w/v) paraformaldehyde for 30 minutes,permeabilized with 0.1% (v/v) Triton X-100, and immunostained withanti-Ras mAb (Cell Biolabs, Inc.). Goat anti-mouse antibodies with AlexaFluor™ 594 (red) were used to visualize the anti-Ras mouse mAb.Immunohistochemistry of treated cells showed not only reduction inprotein levels compared to control cells treated with vehicle (0.1%DMSO), but also a lack of characteristic punctuate pattern of K-Rasdistribution.

Example 6

This example demonstrates the effect of Ras inhibitors on tumor growthin mice.

2×10⁶ H358 human lung cancer cells with mutated K-Ras were implantedsubcutaneously (s.c.) in nude mice. When tumors reached measurable size,10 mg/kg kR-4A-8 or control (DMSO in buffer) was injected s.c. near thetumor every second day for 20 days. No toxicity was detected duringtreatment or necropsy.

Administration of kR-4B-8 abolished the growth of a very aggressivetumor formed by broncheoalveolar carcinoma H358 cells (see FIG. 8).Furthermore, tumors at the time of sacrifice were much smaller intreated (e.g., 0.35 g) versus control (e.g., 1.21 g) mice.

Example 7

This example further demonstrates the effect of Ras inhibitors on tumorgrowth in mice.

Lewis lung carcinoma LLJ2 (LLC1) (ATCC Catalog No. CRL-1642) cells weregrown in DMEM medium containing 10% FBS. Cells were trypsinized andsuspended in PBS. 3×10⁶ LLC1 cells in 200 μl PBS were injectedsubcutaneously to the right flank of 8 weeks old female CB57Bl/6 mice.

Ras inhibitor injections started 2 weeks after LLC1 injections whentumors were at least 3 mm in diameter. For injections, solid kR-H6-48was initially dissolved in DMSO to yield 20 mg/mL stock solution. DMSOstocks were diluted 20-fold in PBS pre-warmed to 35-37° C. 200 μl of theresulting solution (10 mg/kg dose) was injected subcutaneously near thetumor(s) every second day. The control group received 200 μl of 5% DMSOin PBS. Tumor size was measured with a caliper before each injection.The mice were sacrificed when the tumors reached maximal allowed size.

Administration of kR-H6-48 inhibited LLC1 isograft growth in female mice(see FIG. 9).

Example 8

This example demonstrates that Ras inhibitors reduce the amount ofactivated Ras in lung cancer cells.

H358 human lung cancer cells were grown in 6-well plates in DMEM mediumcontaining 10% FBS. When the cells were about 70% confluent, the mediumwas replaced with DMEM containing 1% FBS. Two hours later, Rasinhibitors (kR-H6-48, HR-1, kR-4A-4, or kR-4B-2) were added to provide afinal concentration of 5 μM.

After varying exposure times, the cells were rinsed with PBS and lysed.The lysates were cleared by centrifugation, incubated with immobilizedRaf-RBD beads (which bind Ras) and analyzed by Western blot usingmanufacturer's protocols (Cytoskeleton, Inc., Denver, Colo.; Catalog No.BK008). The bands corresponding to active (GTP-bound) Ras werequantified with the help of MIPAV software.

Administration of the inhibitors reduced the amount of activated(GTP-bound) Ras in lung cancer cells (see FIG. 10).

Example 9

This example demonstrates that Ras inhibitors reduce growth ofRas-dependent cancer cells.

Ras-dependent cancer cells (H358 human lung cancer cells, SKBR3 humanbreast cancer cells, SKOV3 human ovarian cancer cells, ID8 murineovarian cancer cells, and MPR-178 murine prostate cancer cells) weregrown in DMEM medium containing 1% FBS and exposed to varyingconcentrations of Ras inhibitors (kR-H6-48 and HR-1) for 48 hours. Cellgrowth was evaluated utilizing an MTT((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) assay. Theabsorbance of the wells at 544 nm was determined by a FLUOstar/POLARstarGalaxy (BMG Lab Technologies GmbH, Ortenberg, Germany) microplatereader. The assays were performed on untreated (control) and test cells.Cellular responses were calculated from the data using the followingformula: 100×[(T−T₀)/(C−T₀)] for T>T₀ and 100×[(T−T0)/T0] for T<T₀,wherein T₀ corresponds to cell density at the time of drug addition.

Administration of the inhibitors potently inhibited growth ofRas-dependent cancer cells (see FIGS. 11A-B).

Example 10

This experiment demonstrates high affinity binding of lipopeptideanalogs of HVR and helix 6 to recombinant K-Ras.

Recombinant truncated K-Ras protein (1-166) was prepared as described inAbraham et al., Protein Expr. Purif, 73(2): 125-31 (2010). Inhibitorslabeled with fluorescein were dissolved in DMPC/DHPC bicelles (q=2.7,lipids=5% w/v) to an approximate concentration of 1 mg/mL. The exactconcentration was determined by measuring UV absorbance in the range of480-496 nm. Fluorescein extinction coefficient equal to 68,000 M⁻¹cm⁻¹value was used to calculate the exact concentration of the inhibitor.

TABLE 4 Inhibitors used in microscale thermophoresis experiments. SEQCompound ID NO Structure kR-H6-57 87 K(ε-Pal)C(Fluo)FYTLVREIRQYRkR-H6-58 88 Ac-C(Fluo)FYTLVREIRQYR kR-4B-14 89Ac-C(Fluo)KKKKKSKTKK(ε-Pal) kR-4A-11 90 Ac-C(Fluo)KTPGL_(N)VKIKKK(ε-Pal)HR-6 91 Ac-C(Fluo)ESGPGL_(N)L_(N)SL_(N)KK(ε-Pal)

Inhibitors were diluted to an appropriate concentration with bicellessolution. Titration series (16 binding mixtures) were preparedcontaining constant amounts of fluorescent peptide (5 nM for kR-H6-57and 40 nM for other inhibitors) in each sample and varyingconcentrations of recombinant Ras protein. The buffer composition forthe protein dilution contained 25 mM Tris-citrate pH 6.5, 150 mM NaCl,10 mM MgCl₂, 5% Glycerol, 1 mM EDTA, and 1 mM β-mercaptoethanol.Measurements were taken in standard treated capillaries on a MonolithNT. 115 instrument (NanoTemper Technologies GmbH, Germany) using 20%IR-laser power and LED excitation source with λ=470 nm at ambienttemperature. NanoTemper Analysis 1.2.20 software was used to fit thedata and determine the apparent KD values.

Microscale thermophoresis performed in the presence ofmembrane-mimicking micelles and/or bicelles confirmed direct interactionof fluorescent lipopeptide analogs of the HVR and helix 6 withrecombinant truncated GDP-loaded K-Ras (see FIGS. 12 and 13A-D). Theaffinity of inhibitors towards recombinant K-Ras depended on themembrane-mimicking environment and was higher in bicelles than micelles(see FIG. 13A-D).

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A method of inhibiting Ras activity in a cell comprising introducing a peptide or peptidomimetic into the cell, whereby activity of Ras is inhibited; wherein the peptide or peptidomimetic comprises: (a) the amino acid sequence X¹YTLVRX²X³RX⁴X⁵ (SEQ ID NO: 5) or the inverse sequence thereof, wherein the peptide or peptidomimetic comprises about 30 or fewer amino acids; (b) the amino acid sequence KTPGX¹VKIKK (SEQ ID NO: 6) or the inverse sequence thereof, wherein the peptide or peptidomimetic comprises about 30 or fewer amino acids; (c) the amino acid sequence KKSKTK (SEQ ID NO: 7) or the inverse sequence thereof, wherein the peptide or peptidomimetic comprises about 30 or fewer amino acids; (d) the amino acid sequence SGPGX¹X²SX³K (SEQ ID NO: 8) or the inverse sequence thereof, wherein the peptide or peptidomimetic comprises about 30 or fewer amino acids; (e) the amino acid sequence GTQGX¹X²GLP (SEQ ID NO: 9) or the inverse sequence thereof, wherein the peptide or peptidomimetic comprises about 30 or fewer amino acids; (f) eight or more contiguous amino acids of the C-terminal α-helix of a Ras protein or the inverse sequence thereof, wherein the peptide or peptidomimetic comprises a total of about 30 or fewer amino acids; or (g) five or more contiguous amino acids of the hypervariable region (HVR) of a Ras protein or the inverse sequence thereof, wherein the peptide or peptidomimetic comprises a total of about 30 or fewer amino acids.
 2. The method of claim 1, wherein the peptide or peptidomimetic is introduced into the cell by contacting the cell with the peptide or peptidomimetic.
 3. The method of claim 1, wherein the peptide or peptidomimetic is introduced into the cell by contacting the cell with a nucleic acid encoding the peptide or peptidomimetic, whereby the peptide or peptidomimetic is expressed in the cell.
 4. The method of claim 1, wherein the cell is a cancer cell.
 5. The method of claim 1, wherein the cell is in a mammal.
 6. The method of claim 1, wherein the peptide or peptidomimetic comprises the amino acid sequence X¹YTLVRX²X³RX⁴X⁵ (SEQ ID NO: 5) or the inverse sequence thereof, wherein the peptide or peptidomimetic comprises about 30 or fewer amino acids.
 7. The method of claim 1, wherein the peptide or peptidomimetic comprises the amino acid sequence KTPGX¹VKIKK (SEQ ID NO: 6) or the inverse sequence thereof, wherein the peptide or peptidomimetic comprises about 30 or fewer amino acids.
 8. The method of claim 1, wherein the peptide or peptidomimetic comprises the amino acid sequence KKSKTK (SEQ ID NO: 7) or the inverse sequence thereof, wherein the peptide or peptidomimetic comprises about 30 or fewer amino acids.
 9. The method of claim 1, wherein the peptide or peptidomimetic comprises the amino acid sequence SGPGX¹X²SX³K (SEQ ID NO: 8) or the inverse sequence thereof, wherein the peptide or peptidomimetic comprises about 30 or fewer amino acids.
 10. The method of claim 1, wherein the peptide or peptidomimetic comprises the amino acid sequence GTQGX¹X²GLP (SEQ ID NO: 9) or the inverse sequence thereof, wherein the peptide or peptidomimetic comprises about 30 or fewer amino acids.
 11. The method of claim 1, wherein the peptide or peptidomimetic comprises eight or more contiguous amino acids of the C-terminal α-helix of a Ras protein or the inverse sequence thereof, wherein the peptide or peptidomimetic comprises a total of about 30 or fewer amino acids.
 12. The method of claim 1, wherein the peptide or peptidomimetic comprises five or more contiguous amino acids of the hypervariable region (HVR) of a Ras protein or the inverse sequence thereof, wherein the peptide or peptidomimetic comprises a total of about 30 or fewer amino acids.
 19. The method of claim 1, wherein the peptide or peptidomimetic comprises the amino acid sequence of any one of SEQ ID NOs: 10-72 or 75-86, or the inverse sequence thereof.
 13. The method of claim 1, wherein the peptide or peptidomimetic comprises about 20 or fewer amino acids.
 14. The method of claim 1, wherein the peptide or peptidomimetic comprises one or more D-amino acids.
 15. The method of claim 1, wherein the peptide or peptidomimetic further comprises a cell-penetrating motif.
 16. The method of claim 1, wherein the peptide or peptidomimetic further comprises a terminal fatty acid group, an N-terminal palmitoyl residue, an N-terminal myristoyl residue, an N-terminal lauryl residue, or an N-terminal octanoyl residue.
 20. The method of claim 1, wherein the peptide or peptidomimetic interacts with the C-terminal α-helix or Hypervariable Region (HVR) of Ras.
 17. A method for inhibiting the growth or proliferation of a cancer cell comprising administering a peptide or peptidomimetic to the cancer cell, whereby the growth and proliferation of the cancer cell is inhibited; wherein the peptide or peptidomimetic comprises: (a) the amino acid sequence X¹YTLVRX²X³RX⁴X⁵ (SEQ ID NO: 5) or the inverse sequence thereof, wherein the peptide or peptidomimetic comprises about 30 or fewer amino acids; (b) the amino acid sequence KTPGX¹VKIKK (SEQ ID NO: 6) or the inverse sequence thereof, wherein the peptide or peptidomimetic comprises about 30 or fewer amino acids; (c) the amino acid sequence KKSKTK (SEQ ID NO: 7) or the inverse sequence thereof, wherein the peptide or peptidomimetic comprises about 30 or fewer amino acids; (d) the amino acid sequence SGPGX¹X²SX³K (SEQ ID NO: 8) or the inverse sequence thereof, wherein the peptide or peptidomimetic comprises about 30 or fewer amino acids; (e) the amino acid sequence GTQGX¹X²GLP (SEQ ID NO: 9) or the inverse sequence thereof, wherein the peptide or peptidomimetic comprises about 30 or fewer amino acids; (f) eight or more contiguous amino acids of the C-terminal α-helix of a Ras protein or the inverse sequence thereof, wherein the peptide or peptidomimetic comprises a total of about 30 or fewer amino acids; or (g) five or more contiguous amino acids of the hypervariable region (HVR) of a Ras protein or the inverse sequence thereof, wherein the peptide or peptidomimetic comprises a total of about 30 or fewer amino acids.
 18. A method for treating cancer in a host comprising administering to the host a peptide or peptidomimetic, or nucleic acid encoding a peptide or peptidomimetic, whereby cancer is treated; wherein the peptide or peptidomimetic comprises: (a) the amino acid sequence X¹YTLVRX²X²RX⁴X⁵ (SEQ ID NO: 5) or the inverse sequence thereof, wherein the peptide or peptidomimetic comprises about 30 or fewer amino acids; (b) the amino acid sequence KTPGX¹VKIKK (SEQ ID NO: 6) or the inverse sequence thereof, wherein the peptide or peptidomimetic comprises about 30 or fewer amino acids; (c) the amino acid sequence KKSKTK (SEQ ID NO: 7) or the inverse sequence thereof, wherein the peptide or peptidomimetic comprises about 30 or fewer amino acids; (d) the amino acid sequence SGPGX¹X²SX³K (SEQ ID NO: 8) or the inverse sequence thereof, wherein the peptide or peptidomimetic comprises about 30 or fewer amino acids; (e) the amino acid sequence GTQGX¹X²GLP (SEQ ID NO: 9) or the inverse sequence thereof, wherein the peptide or peptidomimetic comprises about 30 or fewer amino acids; (f) eight or more contiguous amino acids of the C-terminal α-helix of a Ras protein or the inverse sequence thereof, wherein the peptide or peptidomimetic comprises a total of about 30 or fewer amino acids; or (g) five or more contiguous amino acids of the hypervariable region (HVR) of a Ras protein or the inverse sequence thereof, wherein the peptide or peptidomimetic comprises a total of about 30 or fewer amino acids.
 19. The method of claim 18, wherein the cancer is colon cancer, pancreatic cancer, leukemia, bladder cancer, salivary gland cancer, melanoma, myeloid malignancy, or germ cell tumors. 